DiMeN Doctoral Training Partnership: Determining the Molecular Mechanism by which Highly Effective, Broadly Neutralising Antibodies are Generated

A major strategy used by pathogens to evade the host immune system is to frequently alter epitopes on their surface proteins. Consequently, new vaccines against common viruses are required every year and the risk of major pandemics is significant. An excellent way for hosts to combat these constantly changing epitopes is to raise broadly neutralising antibodies. These antibodies recognise conserved functional regions of surface proteins and thus offer long term protection against a given pathogen. Whilst humans can generate broadly neutralising antibodies against chronic infections such as malaria, this often takes more than five years and is only apparent in 5-10% of the chronically infected population. Cattle, on the other hand, generate broadly neutralising antibodies against highly evasive pathogens such as HIV within 6 weeks. This project aims to understand the molecular mechanism by which cattle generate broadly neutralising antibodies so efficiently. This has major health and wealth implications: By harnessing the mechanism used by cattle, this pathway could be enhanced in other organisms to enable the production of humanised broadly neutralising antibodies for therapeutic purposes. Moreover, it could allow development of more reliable antibodies for research purposes.

A common feature of broadly neutralising antibody genes is the insertion of a piece of DNA precisely into the variable exon. This increases the length of complementarity determining region 3 (CDR3) of the protein, allowing the antibody to “reach” the conserved regions of pathogen proteins. In humans, these inserted sequences appear to originate from transcribed regions but the origin in cattle, as well as the mechanism of insertion, remains unknown. Cattle must be exposed to antigen before sequence insertion occurs. Following insertion, the sequences become extensively mutated to generate increased antibody diversity. Therefore, to understand the molecular mechanism by which broadly neutralising antibodies are produced, this project will firstly determine the origin of the sequences that are inserted into the antibody genes using a combination of cloning, next generation sequencing and bioinformatics techniques.

Next, it will examine the cell type(s) in which insertion into the antibody gene occurs. Antibody genes are generated via V(D)J recombination in pro-B cells in the bone marrow and become further modified in germinal centres of lymph nodes. The cell type(s) in which inserts occur will be investigated using available bovine antibodies and flow cytometry, together with molecular cloning techniques.
Thirdly, the project will thoroughly investigate the mechanism of DNA insertion. A favoured hypothesis is that RNA is inserted into DNA breaks that is then reverse transcribed during DNA repair. This mechanism will be extensively investigated using available bovine B cell lines, followed by verification in primary cells. Specifically, expression of relevant enzymes will be examined using qPCR and the ability of exogenously added RNAs to be inserted and reverse transcribed will be examined using cell culture and molecular biology techniques.
Finally, the project will examine if the same pathway is present in mouse B cells, also using molecular biology techniques.

Together, these studies will lay the groundwork for producing humanised broadly neutralising antibodies in mouse cells, which has immense therapeutic potential.